构建细胞内环境响应型荧光探针研究纳米载体递药机制

Although nanotechnology still develop rapidly in recent years, the pace of nano-drug researches is slowdown, it indicates that they may confront some grand challenges during their development. The drug delivery mechanism study of nano drug delivery systems (NDDS) are laggard relative to NDDS themselves. Up to now, researches on related mechanism are highly depend on the detection of model drugs or signals of labelled fluorescent/radioactive probes. However, these signals always fail to accurately reflect the in vivo drug delivery behavior of nanocarriers, and free probe signals are frequently mistaken as the performance of nanocarriers. Herin, in this project, we aim to develop an intracellular environment responsive fluorescent probe based on the principles of fluorescent resonance energy transfer (FRET), in which the specific recognition ability of aptamer and reduction of disulfide bond are used to sense the widely distributed adenosine triphosphate (ATP) and glutathione (GSH) intra cell. After an sufficient feasibility study, the probe is loaded into the classic NDDS—liposomes to study their in vivo drug delivery mechanism based on its intracellular reporting response. This project is expected to gain more objective details than current reports, which may enrich the drug delivery theory of liposome and sever as an reference for further research and development. Moreover, this probe can be extended to mechanistic studies of other nanocarriers, with important scientific significance and good application prospect.

近年来虽然纳米科技继续高速发展，但纳米药物的研究却放缓了脚步，这预示着其发展过程中遭遇了重大挑战。与纳米给药系统本身相比，其体内递药机制的研究却相对滞后。目前，机制研究主要依赖对模型药物或所标记荧光/放射探针信号的检测。然而，这些检测信号往往并不能够准确地反映纳米载体的递药行为，游离信号常被误读为载体的行为。本项目拟基于荧光共振能量转移原理，利用核酸适体特异性识别和二硫键还原反应实现探针对细胞内广泛分布的三磷酸腺苷（ATP）和谷胱甘肽（GSH）的灵敏响应，构建细胞内环境响应型荧光探针。在对探针的适用性进行充分研究的基础上，以经典纳米载体脂质体为例，利用该探针对胞内释药行为的报告性响应研究其体内递药机制。有望获得有别于现行报道的更加客观的细节信息，丰富脂质体递药理论，为该类制剂研发提供参考。此外，该探针还可推广应用到其他纳米载体的递药机制研究中，具有重要的科学意义和良好的应用前景。

细胞内释放对于那些不稳定、膜渗透性差特别是靶点在细胞内的药物来说意义重大。纳米载体虽然在药物递送方面展现出独特的优势，但由于其尺寸极小，给药后的体内微观变化过程很难监测。正因为如此，纳米载体胞内递药有效性在开发前期很难评价，从而使纳米制剂临床转化失败率居高不下。.本项目第一部分设计制备了GSH和ATP双响应的荧光增强型探针（以下简称A-QDs）。A-QDs本身呈荧光淬灭状态，然而当其被分散到含GSH或ATP的溶液中或摄取到相关细胞模型中时荧光都会迅速复现。由于细胞内GSH和ATP浓度是细胞外的近千倍，因此A-QDs可用来报告细胞内环境。以常规水溶性荧光染料罗丹明B作对照，分别制备了载A-QDs和罗丹明B的脂质体，在与HepG2细胞共孵育过程中，载A-QDs脂质体在细胞内的量子点信号随着孵育时间延长逐渐增多，符合脂质体逐渐摄取并释放药物的客观规律；而载罗丹明B的脂质体在细胞内的罗丹明B信号始终存在，并没有明显的时间相关性。因此，我们认为A-QDs能作为囊泡型载体胞内递药过程可视化监测的有效工具。.针对胞内递药的重要性，本项目第二部分合成了一个新的含二硫键化学交联剂3,3'-二硫代二丙酰氯, 用该交联剂交联环糊精金属有机框架材料（CD-MOF）后在水中脱去钾离子制得一类新的具有GSH触发降解特性的立方凝胶颗粒（ssCGP）。该颗粒保留了CD-MOF的立方体结构和多孔隙特征，并在正常的细胞培养介质中内稳定性良好，每克可载盐酸阿霉素约45 mg，载阿霉素后该纳米载体在常规介质中4 h的累积释放度小于10 %，而在含二硫键还原剂（如GSH和TCEP等）的介质中可迅速释放完全。鉴于细胞内GSH浓度远远高于细胞外，因此该载体有望成为性质优良的细胞内递药载体.. 综上所述，本项目制备的荧光探针有望为纳米载体细胞内递药效果评价提供有力工具；所制备的可在细胞内迅速降解释药的纳米立方凝胶颗粒有望丰富胞内智能递药系统。